1. Abstract. We synthesized a novel set of non-toxic compounds that kill proliferating and nonproliferating glioma cells residing in perinecrotic tumor microenvironments. These hypoxic glioma populations have increased resistance to conventional radiation therapy coupled with alkylating agents such that cancer recurs in greater than 90% of individuals with high grade gliomas. The design and syntheses of these novel compounds were derived from our recent demonstration that urokinase plasminogen activator (uPA) becomes enzymatically activated intracellularly in hypoxic and acidotic high grade human gliomas. Amiloride is an FDA-approved drug that selectively inhibits uPA, but not other proteases. We synthesized and evaluated a series of amiloride-based compounds that inhibit either total or extracellular uPA activity in human glioma cells. Interestingly, forms of the drugs excluded from the cell interior and act only on surface uPA are cytostatic. Significantly, congeners that permeate the plasma membrane and also inhibit intracellular uPA trigger glioma cell death, yet do not affect normal brain cell types. Our pharmacological findings are consistent with RNA interference experiments demonstrating that inhibition of uPA mRNA initiates apoptosis of glioma cells by unknown cellular mechanisms. These observations point to the novel notion that infiltrative, hypoxic tumor cells can become reliant on intracellular uPA for their survival. Since intracellular uPA activation is not observed in normal tissue cell types or in normal adult brain, these observations suggest that intracellular uPA may represent an important drug target of malignant glioma cells that survive and recur in hypoxic-ischemic microenvironments. The selective anti-glioma cytotoxicities and absence of CNS toxicities of our lead compounds in rat orthotopic glioma xenografts are encouraging given the extremely poor outcome of most individuals having high grade gliomas. Aim 1 will investigate the in vivo efficacies of the lead compounds in a NOD/SCID-gamma murine intracerebral glioma xenograft model. We will investigate the efficacies of our lead compounds on {1} primary glial tumor growth kinetics and on {2} recurrent glioma following radiation therapy and temozolamide (TMZ) treatment. Because uPA inhibition impedes glioma neovascularization, we will also investigate {3} the potential synergism of our lead compounds to prevent or retard the previously described disseminated intracerebral growth of VEGF-depleted glioma cells in mice. Aim 2 will utilize these novel small molecule uPA inhibitors to identify the intracellular mechanisms contributing to their selective anti-glioma cytotoxicity. We will also target total uPA using RNA interference and compare resultant glioma cell death pathways to those identified using our small molecule inhibitors.